Lower alkyl esters of trans-9-oxodec-2-enoic acid



United States Patent National Research Development Corporation, London,

England, a corporation of Great Britain No Drawing. Filed Nov. 30, 1961, Ser. No. 176,170 3 Claims. (Cl. 260-410.9)

This application is a continuation in part of application Serial No. 71,597, filed November 25, 1960, now U.S. Patent 3,162,659, granted December 22, 1964.

The present invention relates to the queen substance of the honeybee (Apis mellifera and A. indica) and active analogues thereof. The queen substance is produced by queen honeybees, and has the effect in the colony of inhibiting the development of ovaries in workers and of inhibiting the rearing of queens by the workers.

Objects of the present invention are to provide synthetic products exhibiting queen substance activity and processes by which they may be prepared.

Although activity attributable to the existence of a queen substance was observed before the present invention in alcoholic extracts of queen bees, attempts to isolate a definite active single substance (which it was considered would be a steroid, a wax or a parafin) in a pure state had been unsuccessful because, ascan now be seen, of difiiculties produced by the royal jelly acid (lO-hydroxy-dec- 2-enoic acid) contained in the crude material.

As stated in co-pending application Serial No. 71,597 of Callow, Butler and Johnston filed November 25, 1960, the recrystallisable substance is trans-9-oxo-dec-2-enoic acid and the present invention is based on the discovery of a synthetic process by which this substance may be provided synthetically as also may its salts and other active functional derivatives especially alkyl esters, for example with alcohols containing from 1 to 4 carbon atoms. The highest melting point found for the pure acid so prepared is 52 -53 C. Prior to this invention, the acid was obtainable only in amounts, too trivial for commercial use, by extraction of queen bees.

In the synthetic process of this invention, a l-alkyl trans-9-oxo-dec-2-enoate, i.e. an alkyl ester of the acid is prepared by reacting a l-monoalkyl trans-non-Z-enedioate acid halide of the general formula XOC.(CH CH:CH.COOR

(in which X represents a halogen atom, preferably chlorine, and R represents an alkyl group) with a dialkyl cadmium or other metal alkyl to yield a complex which is decomposed with water. The acid itself may be obtained from the ester by acid hydrolysis. Attempts to obtain the acid by hydrolysis with alkali hydroxides have been unsuccessful.

As the hydrolysis of the ester present difficulties and as the ester (like other functional derivatives e.g. the amide and substituted amides) is found to be active, it is preferred to employ the ester itself as the final active material.

In a preferred form of the process the l-monoalkyl trans-non-Z-enedioate acid halide is prepared from a 1- alkyl hydrogen trans-non-2-enedioate (by reaction with thionyl chloride or other acid halide-forming agent, e.g. phosphorus trichloride) which may, in accordance with the invention, be produced from a monoalkyl aze'late acid halide by selective halogenation, preferably selective bromination to yield a 9-alkyl 2-haloazela-te-1-acid halide which is then subjected to the steps of esterification, dehydrohalogenation (for which purpose 2,4,6 collidine is a convenient base) and partial hydrolysis. Conveniently the monoalkyl azelate acid halide is monomethyl or monoethyl azelate acid chloride. The former is a known substance which may be prepared from azelaic acid,

HO C(CH CO H via the monomethyl ester thereof.

Given in order, the intermediates involved in the synthesis of trans-9-oxo-dec-2-enoic acid from azelaic acid are:

1. R O2C.(CH2)1.COzH Alkyl hydrogen azelate.

2. R O2C.(GH2)1.COX Nllloilipilkyl azelate acid 3. R O2c.(CHz)a.CHX .COX Q-Alkyl 2-haloazelate 1- acid halide.

4. R 02C.(CHz)u.OHX .CO R Q-AlkKyl; l-alkyl 2-haloaze a e.

5. R O2C.(CHz) .CH:CH.CO R Q-Alkyl l-alkyl transnon-2-enedioate.

6. HOZC.(CHZ) .CH:CH.CO R lzAlkyl hydrogen transnonZenediOate.

7. X OC.(CH2)5.CH:CH.COzR 1-M0noalkyltrans-n0u-2- enedioate Q-acid halide.

8. CH .CO.(OH .CH:CH.CO R l-Alkytl trans-9-oxodec-2- enoa e.

In the formulae, R represents an alkyl group preferably a methyl or ethyl group, R represents an alkyl group containing from 1 to 4 carbon atoms e.g. a methyl or isopropyl group, X and X represent halogen atoms preferably chlorine atoms and X represents a halogen atom, preferably a bromine atom.

Intermediates 1 to 4 are preferably not isolated in carrying out the synthesis.

In the partial hydrolysis of intermediate 5 by the action of alkalis, hydrolysis takes place most readily at the saturated end of the chain. Thus if the partial hydrolysis is carried out using a limited amount of alkali, preferably about 1 mol, the product is a mixture of unhydrolysed ester, hydrolysed ester and free dicarboxylic acid. In the fraction of half hydrolysed ester the desired ester with the ethylenic linkage in the apt-position to the unhy drolysed carbalkoxy-group predominates.

The following example, in which all temperatures are expressed in degrees centigrade, is given by way of illustration:

EXAMPLE Prepa'ration of dialkylbromoazelates from alkyl hydrogen azelates Methyl hydrogen azelate (90.3 g.) was mixed with 140 ml. of thionyl chloride and the mixture heated under reflux on a water-bath for 2 hours. At the end of this time the formation of the acid chloride was complete, and bromination was carried out by addition of 1.05 mols of bromine (78.5 g.) over a period of 4 hours to the gently boiling solution. After allowing to cool and stand overnight the mixture was poured into an excess (300 m1.) of methanol. A vigorous reaction took place, with evolution of sulphur dioxide and hydrogen chloride. After 2 hours, during which the mixture was shaken at intervals, the product was poured into water, the organic layer, which is denser than water, was separated, and the aqueous layer was extracted with ether twice, the ether extracts being added to the organic layer first separated. The ethereal mixture was dried over sodium sulphate, the ether removed by evaporation, and the residue distilled under reduced pressure. Dimethyl bromoazelate is a liquid distilling at 112 0.1 mm. or 134-140 1.5 mm. Yield, 72 g. Analysis.Found: C, 44.6; H, 6.26; Br, 26.5 percent. C H O Br requires C, 44.8; H, 6.5l=Br, 27.1 percent.

By a similar method, but pouring the brominated acid chloride into isopropyl alcohol, there has been obtained methyl isopropyl brornoazelate,

3 B.P. 140-148/1.8 mm. (Found: C, 48.6; H, 7.2; Br, 24.7 percent. C H O Br requires C, 48.3; H, 7.1; Br, 24.7 percent.)

Further, starting with ethyl hydrogen azelate, there has been obtained ethyl isopropyl bromoazelate,

B.P. 151-152/1.8 mm., 11 1.4558. (Found: C, 49.9; H, 7.43; Br, 25.3 percent. C H O Br requires C, 49.9; H, 7.48=Br, 23.7 percent.)

These three dialkyl bromoazelates are all useful intermediates in the synthesis of 9-oxodec-2-enoic acid.

Dialkyl esters of trans-non-Z-enedioic acid Dimethyl bromoazelate (72 g.) was mixed with 2 volumes of 2,4,6-collidine and the mixture was boiled gently under reflux for 1 hour. The product, after cooling, was poured into water. The heavy oil was separated and the aqueous layer extracted twice with ether. The ether extracts were added to the oil and the mixture treated with dilute hydrochloric acid until the reaction to test paper was acid. The organic layer was then dried over sodium sulphate and, after removal of the ether, was distilled under reduced pressure. The product distilled in one main fraction, B.P. 1l7126/0.3 mm. yield 13.5 g. (Found: C, 60.4; H, 8.3 percent. C H O requires C, 61.6=H, 8.5 percent.) The infra red absorption spectrum of the liquid dimethyl trans-non-2-enedioate shows a peak at 1647 cm. characteristic of an ethylenic linkage.

Other esters, also potentially useful as intermediates in the preparation of 9-oxodec-2-enoic acid, may be prepared by analogous methods. Methyl isopropyl trans-non-2- enedioate with the ethylenic linkage in the ali position to the carbopropoxy-group, has B.P. 1l4120/0.3 mm. Ethyl isopropyl trans-non-2-enedioate has B.P. l32136/ 15 mm. (Found: C, 63.9; H, 9.1 percent. C I-1 requires C, 65.6; H, 9.5 percent.)

Mono-alkyl esters of trans-non-Z-enedioic acid Dimethyl trans-non-Z-enedioate (13.5 g.) was treated at 20 with a mixture of 35 ml. of percent by weight aqueous potassium hydroxide solution and 70 ml. of methanol. After standing overnight the mixture was diluted with water and unchanged ester extracted with ester. The aqueous solution was then acidified with hydrochloric acid and again extracted with ether several times. The combined ethereal extracts were dried over sodium sulphate, the ether removed and the residue distilled under reduced pressure. After a small amount of low-boiling material had passed over, the desired half ester came over at 152153/O.3 mm. Yield 4.7 g. The isopropyl ester, B.P. 142148/1.5 mm. or 130l32/0.8 mm. was prepared similarly from the methyl isopropyl and ethyl isopropyl diesters.

M onoalkyl trdns-non-Z-enedioate acid chloride The monomethyl trans-non-Z-enedioate obtained as described (5.6 g.) was mixed with 12 ml. of thionyl chloride and a piece of porous pot added to assist the evolution of gas. After keeping overnight at room temperature the reaction mixture was heated for half an hour on the steam bath, the excess of thionyl chloride was removed at the water-pump, and the residue was distilled under reduced pressure. The acid chloride distilled at 114- 1l6/0.6 mm. Yield 5.1 g.

The analogous isopropyl ester has also been prepared, in a less pure condition, boiling over the range l24-132 1.3 mm.

Methyl trans-9-0x0dec-2-erz0ate A Grignard reagent was prepared from 1.23 g. magnesium and an excess of methyl bromide in ether. To

this was added 4.4. g. of cadmium chloride while the reac- I benzene was added, and then partly distilled off, and finally 5.12 g. of monomethyl trans-non-Z-enedioate acid chloride. A vigorous reaction took place, and after heating on the steam-bath for 10 minutes the mixture was cooled, decomposed with ice and dilute sulphuric acid, and the product extracted with benzene. The benzene extracts were washed with water, 5 percent sodium carbonate solution, again with water and finally dried, the benzene removed, and the residue distilled under reduced pressure. There was obtained 2.22 g. of methyl trans-9-oxo-dec-2- enoate, B.P. ll6l20/1.5 mm.

Isopropyl trans-9-oxodec-2-enoate, B.P. -130/1 mm. was obtained by a similar reaction. Both of the esters were found to possess biological activity in the test for queen substance (inhibition of building of queen cells.)

T rans-9-oxodec-2-en0ic acid This acid was obtained from the isopropyl ester as follows. The ester (0.25 g.) was dissolved in acetone (4 ml.) and concentrated hydrochloric acid (1 ml.) was added. The mixture was kept at room temperature for five days. It was then diluted with water and extracted with ether. The ether solution was extracted with aqueous 5 percent sodium carbonate solution. The alkaline solution was acidified with hydrochloric acid and the trans-9-oxodec-2-enoic acid which separated as an oily product was extracted with ether. The ethereal solution was dried and evaporated. The residue 0.1 g. deposited crystals when kept at 2 C. These crystals separated from the adherent oil, melted at 45-52 and showed the characteristic infra-red absorption of queen substance obtained from queen bees. It also had the same rate of travel on a paper chromatogram as the natural material and, within the limits of error of the bioassay, had quantitatively the same biological activity in inhibiting construction of queen cells.

The bioassay procedure involved providing groups of bees with dead queens which had been ireed from queen substance by extraction and then impregnated by dipping into an alcoholic solution of the material to be tested. Tested in this way the natural material and the synthetic acid both inhibited queen cell production when provided in an amount of about 0.13 g. per bee.

As the products of the invention are active in extremely small amounts they should be used in the form of com positions in which they are associated with a diluent or a carrier, preferably a coherent carrier. From the coherent carrier the bees can take the active material, as from a queen, by licking. Diluents such as liquids or pulverulent solids are of little value as alternatives to coherent carriers for general use.

The compositions have two principal uses, i.e. the reduction of the swarming tendency and favouring the ac' ceptance of a newly introduced queen.

A new queen is usually introduced into the hive in a queen cage, and for use in favouring her acceptance by the colony, the composition takes the form of a coherent solid material through which the bees must penetrate in order to reach her. For this purpose a carrier in the form of paper or sugar candy which may be secured across, or plugged into, the entrance of the cage gives the product a suitable form.

The use of the active material to reduce the swarming tendency, involves supplying it to the colony over a prolonged period, e.g. up to three months, and to this end the carrier should be of such form as to provide a slow release thereof. Convenient compositions are a solid, for example, low molecular weight polyethylene, impregnated with the active material and allowing its slow migration to the surface, or a composition in which the carrier is a wick provided with a reservoir containing the active material in solution. A dead queen, wood, or other material normally found in the hive, soaked in a solution of the active material, may be employed.

the synthetic active material will be a trivial percentage 10 of the value of the colonies and to illustrate the significance of the invention it may be stated that a saving would be achieved even if use of the compositions were to cause a total loss of 15% of the colonies treated.

Iolaim: l. A lower alkyl ester of trans-9-oxodec-2-enoic acid with an alcohol containing from 1 to 4 carbon atoms.

2. Methyl trans-9-0Xodec-2-enoate. 3. Isopropyl trans-9-oxodec-2-enoate.

References Cited by the Examiner C'allow et al.: Bee World 41, 152-153 (1960), cited in Chem. Abstracts 54, 23096d (1960).

Barbier et al.: Compt. Rend. 250, 44674469 (1960), cited in Chem. Abs. 54, 23078i (1960).

Butenandt et a1.: Z. Physiol Chem. 308, 284289 (1957).

CHARLES B. PARKER, Primary Examiner.

15 DANIEL D. HORWITZ, A. H. SUTTO,

Assistant Examiners. 

1. A LOWER ALKYL ESTER OF TRANS-9 OXODEC-2-ENOIC ACID WITH AN ALCOHOL CONTAINING FROM 1 TO 4 CARBON ATOMS. 